Specific cell killing can be essential for the successful treatment of a variety of diseases in mammalian subjects. Typical examples of this are in the treatment of malignant diseases such as sarcomas and carcinomas. However the selective elimination of certain cell types can also play a key role in the treatment of other diseases, especially hyperplastic and neoplastic diseases.
The most common methods of selective treatment are currently surgery, chemotherapy and external beam irradiation. Targeted radionuclide therapy is, however, a promising and developing area with the potential to deliver highly cytotoxic radiation to unwanted cell types. The most common forms of radiopharmaceutical currently authorised for use in humans employ beta-emitting and/or gamma-emitting radionuclides. There has, however, been some interest in the use of alpha-emitting radionuclides in therapy because of their potential for more specific cell killing.
The radiation range of typical alpha emitters in physiological surroundings is generally less than 100 micrometers, the equivalent of only a few cell diameters. This makes these sources well suited for the treatment of tumours, including micrometastases, because they have the range to reach neighbouring cells within a tumour but if they are well targeted then little of the radiated energy will pass beyond the target cells. Thus, not every cell need be targeted but damage to surrounding healthy tissue may be minimised (see Feinendegen et al., Radiat Res 148:195-201 (1997)). In contrast, a beta particle has a range of 1 mm or more in water (see Wilbur, Antibody Immunocon Radiopharm 4: 85-96 (1991)).
The energy of alpha-particle radiation is high in comparison with that carried by beta particles, gamma rays and X-rays, typically being 5-8 MeV, or 5 to 10 times that of a beta particle and 20 or more times the energy of a gamma ray. Thus, this deposition of a large amount of energy over a very short distance gives α-radiation an exceptionally high linear energy transfer (LET), high relative biological efficacy (RBE) and low oxygen enhancement ratio (OER) compared to gamma and beta radiation (see Hall, “Radiobiology for the radiologist”, Fifth edition, Lippincott Williams & Wilkins, Philadelphia Pa., USA, 2000). This explains the exceptional cytotoxicity of alpha emitting radionuclides and also imposes stringent demands on the biological targeting of such isotopes and upon the level of control and study of alpha emitting radionuclide distribution which is necessary in order to avoid unacceptable side effects.
Table 1 below shows the physical decay properties of the alpha emitters so far broadly proposed in the literature as possibly having therapeutic efficacy.
TABLE 1Candidate nuclideT1/2*Clinically tested for225Ac10.0daysleukaemia211At7.2hoursglioblastoma213Bi46minutesleukaemia223Ra11.4daysskeletal metastases224Ra3.66daysankylosing spondylitis*Half life
So far, with regards to the application in radioimmunotherapy the main attention has been focused on 211At, 213Bi and 225Ac and these three nuclides have been explored in clinical immunotherapy trials.
Several of the radionuclides which have been proposed are short-lived, i.e. have half lives of less than 12 hours. Such a short half-life makes it difficult to produce and distribute radiopharmaceuticals based upon these radionuclides in a commercial manner. Administration of a short-lived nuclide also increases the proportion of the radiation dose which will be emitted in the body before the target site is reached.
The recoil energy from alpha-emission will in many cases cause the release of daughter nuclides from the position of decay of the parent. This recoil energy is sufficient to break many daughter nuclei out from the chemical environment which may have held the parent, e.g. where the parent was complexed by a ligand such as a chelating agent. This will occur even where the daughter is chemically compatible with, i.e. complexable by, the same ligand. Equally, where the daughter nuclide is a gas, particularly a noble gas such as radon, or is chemically incompatible with the ligand, this release effect will be even greater. When daughter nuclides have half-lives of more than a few seconds, they can diffuse away into the blood system, unrestrained by the complexant which held the parent. These free radioactive daughters can then cause undesired systemic toxicity.
The use of Thorium-227 (T1/2=18.7 days) under conditions where control of the 223Ra daughter isotope is maintained was proposed a few years ago (see WO 01/60417 and WO 02/05859). This was in situations where a carrier system is used which allows the daughter nuclides to be retained by a closed environment. In one case, the radionuclide is disposed within a liposome and the substantial size of the liposome (as compared to recoil distance) helps retain daughter nuclides within the liposome. In the second case, bone-seeking complexes of the radionuclide are used which incorporate into the bone matrix and therefore restrict release of the daughter nuclides. These are potentially highly advantageous methods, but the administration of liposomes is not desirable in some circumstances and there are many diseases of soft tissue in which the radionuclides cannot be surrounded by a mineralised matrix so as to retain the daughter isotopes.
More recently, it was established that the toxicity of the 223Ra daughter nuclei released upon decay of 227Th could be tolerated in the mammalian body to a much greater extent than would be predicted from prior tests on comparable nuclei. In the absence of the specific means of retaining the radium daughters of thorium-227 discussed above, the publicly available information regarding radium toxicity made it clear that it was not possible to use thorium-227 as a therapeutic agent since the dosages required to achieve a therapeutic effect from thorium-227 decay would result in a highly toxic and possibly lethal dosage of radiation from the decay of the radium daughters, i.e. there is no therapeutic window.
WO 04/091668 describes the unexpected finding that a therapeutic treatment window does exist in which a therapeutically effective amount of a targeted thorium-227 radionuclide can be administered to a subject (typically a mammal) without generating an amount of radium-223 sufficient to cause unacceptable myelotoxicity. This can therefore be used for treatment and prophylaxis of all types of diseases at both bony and soft-tissue sites.
In view of the above developments, it is now possible to employ alpha-emitting thorium-227 nuclei in endoradionuclide therapy without lethal myelotoxicity resulting from the generated 223Ra. Nonetheless, the therapeutic window remains relatively narrow and it is in all cases desirable to administer no more alpha-emitting radioisotope to a subject than absolutely necessary. Useful exploitation of this new therapeutic window would therefore be greatly enhanced if the alpha-emitting thorium-227 nuclei could be complexed and targeted with a high degree of reliability.
Because radionuclides are constantly decaying, the time spent handling the material between isolation and administration to the subject is of great importance. It would also be of considerable value if the alpha-emitting thorium nuclei could be complexed, targeted and/or administered in a form which was quick and convenient to prepare, preferably requiring few steps, short incubation periods and/or temperatures not irreversibly affecting the properties of the targeting entity. Furthermore, processes which can be conducted in solvents that do not need removal before administration (essentially in aqueous solution) have the considerable advantage of avoiding a solvent evaporation or dialysis step. This reduces the time and complexity of preparation, which is of key significance in the generation of radiopharmaceuticals, which decay continuously to contaminant daughter products.
In view of the need for selectivity in the delivery of cytotoxic agents in therapy, there is an evident need for targeting of alpha-radionuclide complexes. However, conjugates of suitable chelators with a small targeting peptide or small protein tend to show poor solubility in aqueous systems because the small biomolecule cannot keep the insoluble chelate in solution. Poor solubility leads to aggregation and precipitation. Aggregates are unacceptable in a drug preparation to be administered to human subjects and evidently precipitation renders a composition entirely unusable.
Furthermore, also with a larger targeting peptide/protein, such as a monoclonal antibody, the chelator will be exposed on the surface of the conjugate as a hydrophobic ‘spot’. This might in some contexts lead to issues with micro aggregation.
In a biological system, such as in a human patient, hydrophobicity in general is associated with undesirable uptake in the liver. Evidently this is much more serious with highly cytotoxic agents such as alpha-emitters than for typical drug compounds. Hydrophobicity of the chelator also increases the risk of an immune response, as hydrophobicity facilitates stronger binding of antibodies produced by the host's immune system. Again this is of particular concern with alpha-emitters due to their exceptional cytotoxicity. There is thus evidently a considerable need of methods for the selective delivery of alpha-emitting thorium radionuclides by conjugates having increased hydrophilicity, particularly of the ligand portion, so as to address one or more of the issues discussed above
Octadentate chelating agents containing hydroxypyridinone groups have previously been shown to be suitable for coordinating the alpha emitter thorium-277, for subsequent attachment to a targeting moiety (WO2011098611). Octadentate chelators were described, containing four 3,2-hydroxypyridinone groups joined by linker groups to an amine-based scaffold, having a separate reactive group used for conjugation to a targeting molecule. Preferred structures of the previous invention contained 3,2-hydroxypyridinone groups having a methyl substituted nitrogen in position 1 of the heterocyclic ring, and were linked to the amine based scaffold by a an amine bond involving an formic acid attached at position 4, as shown in by the compounds ALG-DD-NCS, ALG1005-38, Bb-1-HOPO-1-DEBN. In the experiment where one of these hydroxypyiridinone containing molecules was conjugated to a tumor targeting antibody, the molecule was dissolved in the organic solvent DMSO since it could not be dissolved in aqueous buffers.
The use of specific targeting moieties in cytotoxic therapy (such as cancer chemotherapy or endoradionuclide therapy) is now a well established method and numerous targets and potential targets are known. These are typically cell-surface or matrix markers (such as receptors) which are to some extent preferentially expressed in diseased cells or in cells associated with diseased cells, or in the nearby matrix. Specific binding moieties can serve to target, carry and/or bind cytotoxic elements (such as chemical toxins or radionuclides) to the vicinity of an unwanted cell type (e.g. neoplastic cells) and thereby improve the selectivity of cell killing provided by the cytotoxic agent. In order to take advantage of such specific binding property, the binding moiety must be conjugated or conjugatable to the cytotoxic agent (such as the complexed radionuclide). Many cell surface and matrix targets are known including receptors such as folate binding receptor, CD22, CD33, estrogen and progesterone receptors and many others. Typically an antibody, antibody fragment or smaller binding molecule (such as an “affibody”) is generated with specificity for such a cell surface marker and conjugated to the cytotoxic agent. Any of these known methods and markers are potentially usable with radionuclide agents. However, preparation ease and time is important and for smaller binders, solubility can be critical because the conjugate as a whole must be soluble for administration.
The present inventors have now unexpectedly established that the use of a 4+ thorium-227 ion complexed by an octadentate hydroxypyridinone (HOPO)-type ligand comprising four HOPO moieties of which at least one is substituted with a suitable solubilising moiety can provide a dramatic improvement in solubility and corresponding properties of the complex.